bims-novged Biomed News
on Non-viral vectors for gene delivery
Issue of 2024–04–21
seven papers selected by
the Merkel lab, Ludwig-Maximilians University
  1. Microfluidic Platform Enables Shearless Aerosolization of Lipid Nanoparticles for mRNA Inhalation.
  2. Targeting Recycling Endosomes to Potentiate mRNA Lipid Nanoparticles.
  3. Elucidating Structural Configuration of Lipid Assemblies for mRNA Delivery Systems.
  4. Recent Progress of Small Interfering RNA Delivery on the Market and Clinical Stage.
  5. Dual-responsive nanocarriers for efficient cytosolic protein delivery and CRISPR-Cas9 gene therapy of inflammatory skin disorders.
  6. Stability indicating ion-pair reversed-phase liquid chromatography method for modified mRNA.
  7. Coarse-grained modeling and dynamics tracking of nanoparticles diffusion in human gut mucus.
  1. ACS Nano. 2024 Apr 15.
    Microfluidic Platform Enables Shearless Aerosolization of Lipid Nanoparticles for mRNA Inhalation.
    Jeonghwan Kim, Antony Jozić, Elissa Bloom, Brian Jones, Michael Marra, Namratha Turuvekere Vittala Murthy, Yulia Eygeris, Gaurav Sahay.
      Leveraging the extensive surface area of the lungs for gene therapy, the inhalation route offers distinct advantages for delivery. Clinical nebulizers that employ vibrating mesh technology are the standard choice for converting liquid medicines into aerosols. However, they have limitations when it comes to delivering mRNA through inhalation, including severe damage to nanoparticles due to shearing forces. Here, we introduce a microfluidic aerosolization platform (MAP) that preserves the structural and physicochemical integrity of lipid nanoparticles, enabling safe and efficient delivery of mRNA to the respiratory system. Our results demonstrated the superiority of the MAP over the conventional vibrating mesh nebulizer, as it avoided problems such as particle aggregation, loss of mRNA encapsulation, and deformation of the nanoparticle morphology. Notably, aerosolized nanoparticles generated by the microfluidic device led to enhanced transfection efficiency across various cell lines. In vivo experiments with mice that inhaled these aerosolized nanoparticles revealed successful lung-specific mRNA transfection without observable signs of toxicity. This MAP may represent an advancement for the pulmonary gene therapy, enabling precise and effective delivery of aerosolized nanoparticles.
    Keywords:  aerosol; gene delivery; lung targeting; mRNA; nebulizer; pulmonary delivery
    DOI:  https://doi.org/10.1021/acsnano.4c00768
  2. Nano Lett. 2024 Apr 19.
    Targeting Recycling Endosomes to Potentiate mRNA Lipid Nanoparticles.
    Jeehae Shin, Cameron J Douglas, Shanwen Zhang, Ciaran P Seath, Huan Bao.
      mRNA lipid nanoparticles (LNPs) have emerged as powerful modalities for gene therapies to control cancer and infectious and immune diseases. Despite the escalating interest in mRNA-LNPs over the past few decades, endosomal entrapment of delivered mRNAs vastly impedes therapeutic developments. In addition, the molecular mechanism of LNP-mediated mRNA delivery is poorly understood to guide further improvement through rational design. To tackle these challenges, we characterized LNP-mediated mRNA delivery using a library of small molecules targeting endosomal trafficking. We found that the expression of delivered mRNAs is greatly enhanced via inhibition of endocytic recycling in cells and in live mice. One of the most potent small molecules, endosidine 5 (ES5), interferes with recycling endosomes through Annexin A6, thereby promoting the release and expression of mRNA into the cytoplasm. Together, these findings suggest that targeting endosomal trafficking with small molecules is a viable strategy to potentiate the efficacy of mRNA-LNPs.
    Keywords:  Lipid nanoparticles; cellular uptake; endosomal recycling; mRNA; mRNA delivery
    DOI:  https://doi.org/10.1021/acs.nanolett.3c04415
  3. ACS Nano. 2024 Apr 19.
    Elucidating Structural Configuration of Lipid Assemblies for mRNA Delivery Systems.
    Hyunhyuk Tae, Soohyun Park, Li Yang Tan, Chungmo Yang, Yong-An Lee, Younghwan Choe, Torsten Wüstefeld, Sangyong Jung, Nam-Joon Cho.
      The development of mRNA delivery systems utilizing lipid-based assemblies holds immense potential for precise control of gene expression and targeted therapeutic interventions. Despite advancements in lipid-based gene delivery systems, a critical knowledge gap remains in understanding how the biophysical characteristics of lipid assemblies and mRNA complexes influence these systems. Herein, we investigate the biophysical properties of cationic liposomes and their role in shaping mRNA lipoplexes by comparing various fabrication methods. Notably, an innovative fabrication technique called the liposome under cryo-assembly (LUCA) cycle, involving a precisely controlled freeze-thaw-vortex process, produces distinctive onion-like concentric multilamellar structures in cationic DOTAP/DOPE liposomes, in contrast to a conventional extrusion method that yields unilamellar liposomes. The inclusion of short-chain DHPC lipids further modulates the structure of cationic liposomes, transforming them from multilamellar to unilamellar structures during the LUCA cycle. Furthermore, the biophysical and biological evaluations of mRNA lipoplexes unveil that the optimal N/P charge ratio in the lipoplex can vary depending on the structure of initial cationic liposomes. Cryo-EM structural analysis demonstrates that multilamellar cationic liposomes induce two distinct interlamellar spacings in cationic lipoplexes, emphasizing the significant impact of the liposome structures on the final structure of mRNA lipoplexes. Taken together, our results provide an intriguing insight into the relationship between lipid assembly structures and the biophysical characteristics of the resulting lipoplexes. These relationships may open the door for advancing lipid-based mRNA delivery systems through more streamlined manufacturing processes.
    Keywords:  biophysics; cationic liposome; cryo-assembly; lipid assembly; lipoplex; mRNA delivery; multilamellar structure
    DOI:  https://doi.org/10.1021/acsnano.4c00587
  4. Mol Pharm. 2024 Apr 17.
    Recent Progress of Small Interfering RNA Delivery on the Market and Clinical Stage.
    Fan Guo, Yan Li, Wenjun Yu, Yuanlei Fu, Jing Zhang, Haiqiang Cao.
      Small interfering RNAs (siRNAs) are promising therapeutic strategies, and five siRNA drugs have been approved by the Food and Drug Administration (FDA) and the European Commission (EC). This marks a significant milestone in the development of siRNA for clinical applications. The approved siRNA agents can effectively deliver siRNAs to the liver and treat liver-related diseases. Currently, researchers have developed diverse delivery platforms for transporting siRNAs to different tissues such as the brain, lung, muscle, and others, and a large number of siRNA drugs are undergoing clinical trials. Here, these delivery technologies and the latest advancements in clinical applications are summarized, and this Review provides a concise overview of the strategies employed for siRNA delivery to both hepatic and extrahepatic tissues.
    Keywords:  delivery platforms; extrahepatic targeting; gene therapy; hepatic targeting; small interfering RNAs
    DOI:  https://doi.org/10.1021/acs.molpharmaceut.3c01158
  5. Sci Adv. 2024 Apr 19. 10(16): eadl4336
    Dual-responsive nanocarriers for efficient cytosolic protein delivery and CRISPR-Cas9 gene therapy of inflammatory skin disorders.
    Echuan Tan, Tao Wan, Qi Pan, Jianan Duan, Song Zhang, Ruijue Wang, Peng Gao, Jia Lv, Hui Wang, Dali Li, Yuan Ping, Yiyun Cheng.
      Developing protein drugs that can target intracellular sites remains a challenge due to their inadequate membrane permeability. Efficient carriers for cytosolic protein delivery are required for protein-based drugs, cancer vaccines, and CRISPR-Cas9 gene therapies. Here, we report a screening process to identify highly efficient materials for cytosolic protein delivery from a library of dual-functionalized polymers bearing both boronate and lipoic acid moieties. Both ligands were found to be crucial for protein binding, endosomal escape, and intracellular protein release. Polymers with higher grafting ratios exhibit remarkable efficacies in cytosolic protein delivery including enzymes, monoclonal antibodies, and Cas9 ribonucleoprotein while preserving their activity. Optimal polymer successfully delivered Cas9 ribonucleoprotein targeting NLRP3 to disrupt NLRP3 inflammasomes in vivo and ameliorate inflammation in a mouse model of psoriasis. Our study presents a promising option for the discovery of highly efficient materials tailored for cytosolic delivery of specific proteins and complexes such as Cas9 ribonucleoprotein.
    DOI:  https://doi.org/10.1126/sciadv.adl4336
  6. J Pharm Biomed Anal. 2024 Apr 10. pii: S0731-7085(24)00184-5. [Epub ahead of print]245 116144
    Stability indicating ion-pair reversed-phase liquid chromatography method for modified mRNA.
    Jonathan Currie, Jacob R Dahlberg, Ester Lundberg, Linda Thunberg, Jonas Eriksson, Fritz Schweikart, Gunilla A Nilsson, Eivor Örnskov.
      Modified messenger RNA (mRNA) represents a rapidly emerging class of therapeutic drug product. Development of robust stability indicating methods for control of product quality are therefore critical to support successful pharmaceutical development. This paper presents an ion-pair reversed-phase liquid chromatography (IP-RPLC) method to characterise modified mRNA exposed to a wide set of stress-inducing conditions, relevant for pharmaceutical development of an mRNA drug product. The optimised method could be used for separation and analysis of large RNA, sized up to 1000 nucleotides. Column temperature, mobile phase flow rate and ion-pair selection were each studied and optimised. Baseline separations of the model RNA ladder sample were achieved using all examined ion-pairing agents. We established that the optimised method, using 100 mM Triethylamine, enabled the highest resolution separation for the largest fragments in the RNA ladder (750/1000 nucleotides), in addition to the highest overall resolution for the selected modified mRNA compound (eGFP mRNA, 996 nucleotides). The stability indicating power of the method was demonstrated by analysing the modified eGFP mRNA, upon direct exposure to heat, hydrolytic conditions and treatment with ribonucleases. Our results showed that the formed degradation products, which appeared as shorter RNA fragments in front of the main peak, could be well monitored, using the optimised method, and the relative stability of the mRNA under the various stressed conditions could be assessed.
    Keywords:  Chemical degradation; Degradation study; Ion-pair reversed-phase liquid chromatography; Modified mRNA; Nucleotides; RNA ladder; Ribonucleic acid; Stability indicating
    DOI:  https://doi.org/10.1016/j.jpba.2024.116144
  7. Int J Biol Macromol. 2024 Apr 11. pii: S0141-8130(24)02239-6. [Epub ahead of print] 131434
    Coarse-grained modeling and dynamics tracking of nanoparticles diffusion in human gut mucus.
    Liming Zhao, Sandra L Arias, Warren Zipfel, Ilana L Brito, Jingjie Yeo.
      The gastrointestinal (GI) tract's mucus layer serves as a critical barrier and a mediator in drug nanoparticle delivery. The mucus layer's diverse molecular structures and spatial complexity complicates the mechanistic study of the diffusion dynamics of particulate materials. In response, we developed a bi-component coarse-grained mucus model, specifically tailored for the colorectal cancer environment, that contained the two most abundant glycoproteins in GI mucus: Muc2 and Muc5AC. This model demonstrated the effects of molecular composition and concentration on mucus pore size, a key determinant in the permeability of nanoparticles. Using this computational model, we investigated the diffusion rate of polyethylene glycol (PEG) coated nanoparticles, a widely used muco-penetrating nanoparticle. We validated our model with experimentally characterized mucus pore sizes and the diffusional coefficients of PEG-coated nanoparticles in the mucus collected from cultured human colorectal goblet cells. Machine learning fingerprints were then employed to provide a mechanistic understanding of nanoparticle diffusional behavior. We found that larger nanoparticles tended to be trapped in mucus over longer durations but exhibited more ballistic diffusion over shorter time spans. Through these discoveries, our model provides a promising platform to study pharmacokinetics in the GI mucus layer.
    Keywords:  Gastrointestinal mucus; Molecular dynamics modeling; Nanoparticle diffusion
    DOI:  https://doi.org/10.1016/j.ijbiomac.2024.131434
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